The Study Of The Effect Of Proteasome Inhibitors On Ovarian Cancer Cell

Objective:Most ovarian cancer share poor prognosis and the highest mortality in female genital tract malignancies because of non-typical early symptoms,lack of early diagnosis and fast disease progression.Among all histological types of the primary ovarian cancer,epithelial ovarian cancer accounts for 70%-90% and thus becomes the most common type.To investigate the possible mechanism of proteasome inhibitor Lactacystin,we detected the vitro cell proliferation rate,survival rate and the related signal level change in ovarian cancer SKOV3 cells.Eventually,we hope to come up with new theoretical ideas and research direction for ovarian cancer treatment,so that the patients can benefit from the reduction of ovarian cancer mortality rate.Methods:At the cellular level,we use the methods of MTT staining and flow cytometry to detect the effects of the proteasome inhibitor Lactacystin for ovarian cancer SKOV3 cells in aspects of growth,proliferation and apoptosis.In the protein and gene level,expression levels of protein and gene were detected by means of Western Blot and RT-PCR to show the changes of cell injury-related signals following injury caused by LAC.Results:(1)Morphological changes of cells under microscope: In the control group,the SKOV3 cells grew well with full cellular morphology,clear boundary,and close connection,which were polygonal or spindle shaped and adhered to the wall.Along with increasing concentrations of Lactacystin,The morphology of SOKV3 cells has gradually changed from polygonal to oval or round.The boundary between cells was blurred and the sense of particle was enhanced,which lead to a poor light transmittance.Also the inactive floating cells increased significantly.The higher LAC concentration we had used,the more obvious phenomenon we observed.(2)MTT results: compared with the control group,the proliferation rate of SKOV3 cells in LAC groups was significantly decreased and the difference was statistically significant,p<0.05.Respectively,the LAC 0.5umol/L group proliferation rate decreased to 65.5±0.10%,LAC 1.0umol/L group proliferation rate decreased to 54.4±0.11%,LAC 2.5umol/L group proliferation rate decreased to 42.5±0.09%,LAC 5.0umol/L group proliferation rate decreased to 29.2±0.12% and LAC 10.0umol/L group proliferation rate decreased to18.9±0.13%.(3)The test results of flow cytometry: Lactacystin could induce the apoptosis of SKOV3 cells in comparison with the control group,and the higher LAC concentration resulted in a higher apoptosis rate in unit time.We observed that there was an obvious concentration dependence of apoptosis rate which is positively related to the drug concentration The difference was statistically significant,P <0.05.(4)Western Blot test results: while the concentration of LAC increased,the expression of Bax protein increased rapidly,and the expression of Bcl-2 protein decreased gradually.The difference was statistically significant,P <0.05.(5)RT-PCR electrophoresis results: along with the increasing LAC concentration,the expression of Bax m RNA increased rapidly,and the expression of Bcl-2 m RNA decreased gradually in equal time,The difference was statistically significant,P <0.05.Conclusion:Proteasome inhibitors LAC can inhibit proliferation and induce apoptosis in ovarian cancer SKOV3 cells.The inference is that the proteasome inhibitor Lactacystin inhibits the activity of ubiquitin-proteasome system,which result in an imbalance of expression of the apoptosis related protein and gene like Bax,bcl-2,etc,and then effect the mitochondrial pathway to induces apoptosis in ovarian cancer SKOV3 cells.